Flagellar incorporation of proteins follows at least two different routes in trypanosomes.

BACKGROUND INFORMATION: Eukaryotic cilia and flagella are sophisticated organelles composed of several hundreds of proteins that need to be incorporated at the right time and the right place during assembly. RESULTS: Two methods were used to investigate this process in the model protist Trypanosoma brucei: inducible expression of epitope-tagged labelled proteins and fluorescence recovery after photobleaching of fluorescent fusion proteins. This revealed that skeletal components of the radial spokes (RSP3), the central pair (PF16) and the outer dynein arms (DNAI1) are incorporated at the distal end of the growing flagellum. They display low or even no visible turnover in mature flagella, a finding further confirmed by monitoring a heavy chain of the outer dynein arm. In contrast, the membrane-associated protein arginine kinase 3 (AK3) showed rapid turnover in both growing and mature flagella, without particular polarity and independently of intraflagellar transport. CONCLUSION: These results demonstrate different modes of incorporation for structural and membrane-associated proteins in flagella. SIGNIFICANCE: The existence of two distinct modes for incorporation of proteins in growing flagella suggests the existence of different targeting machineries. Moreover, the absence of turnover of structural elements supports the view that the length of the mature flagellum in trypanosomes is not modified after assembly.

Novel Broccoli Sulforaphane-Based Analogues Inhibit the Progression of Pancreatic Cancer without Side Effects.

The naturally occurring isothiocyanate sulforaphane, found in Brassicaceae vegetables, is promising in cancer treatment, e.g., by the normalization of enhanced levels of NF-κB-signaling in tumor stem cells. We chemically synthesized seven sulforaphane analogues by substitution of the sulfinyl group (S(O)) to either sulfimidoyl (S(NR)) or sulfonimidoyl (S (O) (NR)) groups, and characterized them in the cell lines of pancreatic cancer and several other tumor entities, including the NCI-60 cell panel. MTT and colony forming assays, flow cytometry, immunohistochemistry, microRNA arrays, bioinformatics, tumor xenotransplantation, and Kaplan Meier survival curves were performed. Compared to sulforaphane, the analogue SF102 was most efficient in inhibition of viability, colony formation, tumor growth, and the induction of apoptosis, followed by SF134. Side effects were not observed, as concluded from the body weight and liver histology of chick embryos and survival of C. elegans nematodes. Among 6659 differentially regulated microRNAs, miR29b-1-5p, and miR-27b-5p were downregulated by sulforaphane compared to controls, but upregulated by SF102 and SF134 compared to sulforaphane, suggesting differential signaling. Each substance was involved in the regulation of several NF-κB-related target genes. In conclusion, sulforaphane analogues are promising for the development of highly active new drugs in cancer treatment.

Novel plant microRNAs from broccoletti sprouts do not show cross-kingdom regulation of pancreatic cancer.

Food-derived plant microRNAs are suggested to control human genes by "cross-kingdom" regulation. We examined microRNAs in sprouts from Brassica rapa sylvestris, known as broccoletti, which are widely used as sulforaphane supplements, and assessed their influence on pancreatic cancer. RNA was isolated from 4-day-old sprouts, followed by deep sequencing and bioinformatic analysis. We identified 2 new and 745 known plant microRNA sequences in the miRbase database and predicted 15,494 human target genes and 76,747 putative 3'-UTR binding sites in these target genes. The most promising candidates were the already known microRNA sequence bra-miR156g-5p and the new sequence Myseq-330, both with predicted human target genes related to apoptosis. The overexpression of the respective oligonucleotides by lipofection did not alter the viability, apoptosis, clonogenicity, migration or associated protein expression patterns in pancreatic cancer cells. These data demonstrate that broccoletti sprouts contain microRNA sequences with putative binding sites in human genes, but the sequences evaluated here did not affect cancer growth. Our database of broccoletti-derived microRNA sequences provides a valuable tool for future analysis.

Inhibition of miR30a-3p by sulforaphane enhances gap junction intercellular communication in pancreatic cancer.

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.

Sulforaphane Induces miR135b-5p and Its Target Gene, RASAL2, thereby Inhibiting the Progression of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal tumors, with poor therapeutic options in the advanced state. The broccoli-derived anti-inflammatory agent sulforaphane was shown to inhibit the progression of pancreatic cancer and other tumor entities. We examined the involvement of pancreatic cancer cell lines were evaluated by microRNA and gene expression arrays, bioinformatics, in silico analysis, qRT-PCR, western blotting, immunohistochemistry, in situ hybridization, self-renewal and differentiation assays, and in vivo xenograft studies. We selected the top nine differentially expressed microRNAs, and miR135b-5p was chosen as the most important candidate for the sulforaphane-induced upregulation of the tumor suppressor gene RASAL2. The expression of miR135b-5p and RASAL2 was almost absent in malignant pancreatic tissues and cell lines, but not in their normal counterparts. Lipofection of miR135b-5p enhanced RASAL2 expression and inhibited ERK1/2 signaling, viability, self-renewal capacity, and tumor growth. These results indicate that miR135b-5p acts as a tumor suppressor via the induction of RASAL2 in PDA.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

Pediatric acute lymphoblastic leukemia-Conquering the CNS across the choroid plexus.

Despite the high prevalence of central nervous system (CNS) involvement in relapsing pediatric acute lymphoblastic leukemia (ALL), our understanding of CNS invasion is still vague. As lymphoblasts have to overcome the physiological blood-CNS barriers to enter the CNS, we investigated the cellular interactions of lymphoblasts with the choroid plexus (CP) epithelium of the blood-cerebrospinal fluid barrier (BCSFB). Both a precurser B cell ALL (pB-ALL) cell line (SD-1) and a T cell ALL (T-ALL) cell line (P12-Ishikawa) were able to actively cross the CP epithelium in a human in vitro model. We could illustrate a transcellular and (supposedly) paracellular transmigration by 3-dimensional immunofluorescence microscopy as well as electron microscopy. Chemotactic stimulation with CXCL12 during this process led to a significantly increased transmigration and blocking CXCL12/CXCR4-signaling by the CXCR4-inhibitor AMD3100 inhibited this effect. However, CXCR4 expression in primary ALL samples did not correlate to CNS disease, indicating that CXCR4-driven CNS invasion across the BCSFB might be a general property of pediatric ALL. Notably, we present a unique in vitro BCSFB model suitable to study CNS invasion of lymphoblasts in a human setting, providing the opportunity to investigate experimental variables, which may determine CNS disease childhood ALL.

The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.